AFFINITY AND SPECIFICITY OF SERINE ENDOPEPTIDASE PROTEIN INHIBITOR INTERACTIONS - EMPIRICAL FREE-ENERGY CALCULATIONS BASED ON X-RAY CRYSTALLOGRAPHIC STRUCTURES

An empirical function was used to calculate free energy change (ΔG) of complex formation between the following inhibitors and enzymes: Kunitz inhibitor (BPTI) with trypsin, trypsinogen and kallikrein; turkey ovomucoid 3rd domain (OMTKY3) with α-chymotrypsin and the Streptomyces griseus protease B; the potato chymotrypsin inhibitor with the protease B; and the barley chymotrypsin inhibitor and eglin-c with subtilisin and thermitase. Using X-ray coordinates of the nine complexes, we estimated the contributions that hydrophobic effect, electrostatic interactions and side-chain conformational entropy make towards the stability of the complexes. The calculated ΔG values showed good agreement with the experimentally measured ones, the only exception being the kallikrein/BPTI complex whose X-ray structure was solved at an exceptionally low pH. In complexes with different enzymes, the same inhibitor residues contributed identically towards complex formation (ΔC(residue) Spearman rank correlation coefficient 0.7 to 1.0). The most productive enzyme-contacting residues in OMTKY3, eglin-c, and the chymotrypsin inhibitors were found in analogous positions on their respective binding loops; thus, our calculations identified a functional (energetic) motif that parallels the well-known structural similarity of the binding loops. The ΔG values calculated for BPTI complexed with trypsin (-21.7 kcal) and trypsinogen (-23.4 kcal) were similar and close to the experimental ΔG value of the trypsin/BPTI complex (-18.1 kcal), lending support to the suggestion that the 107 difference in the observed stabilities (K(A)) of these two complexes reflects the energetic cost of conformational changes induced in trypsinogen during the pre-equilibrium stages of complex formation. In almost all of the complexes studied, the stabilization free energy contributed by the inhibitors was larger than that donated by the enzymes. In the trypsin-BPTI complex, the calculated ΔG contribution of the amino group from the BPTI residue Lys15(9.7 kcal) was somewhat higher than that arrived at in experiments with semisynthetic inhibitor analogs (7.5 kcal). In OMTKY3, different binding loop residues are known to affect differently the binding (ΔΔG) to α-chymotrypsin and protease B; a good qualitative agreement was found between the calculated ΔG(residue) estimates and the experimental ΔΔG data (correlation coefficient 0.7). Large variations were observed in local surface complementarity and related interfacial volume in the two OMTKY3 complexes (by 20 to 60% for some side-chains). Binding surfaces of several inhibitors and enzymes were found to be rich in amino acids with side-chains that incur no conformational entropic penalty on complex formation (Pro, Ala, Gly; non-entropic side-chains). Thus, binding surfaces of these molecules show a composition different from that of antibodies (where aromatic and polar side-chains predominate), suggesting that inhibitors, enzymes and antibodies, in their respective complexes, use different strategies of harnessing free energy of binding. © 1993 Academic Press Limited.